The early events of innate immunity in defense against foreign pathogens remain to be fully understood. DNA sensors are essential stimulators of innate immunity, many of which have only recently begun to be characterized. Defects in immune responses have been linked to numerous prominent human diseases; for example, misregulation of the sensing pathway can trigger autoimmune diseases. Despite the ever-growing number of DNA sensors discovered, all but one have been shown to act exclusively in the cytoplasm of cells. However, several viruses are able to protect their viral genomes throughout the cytoplasm and then deposit this foreign DNA into host cell nuclei. Thus, the discovery and characterization of nuclear DNA sensing provides critical insight into host defense mechanisms during viral infection. Recent studies from the Cristea laboratory and others have established that two PYHIN proteins, IFI16 and AIM2, are DNA sensors during viral infections. The hallmark of these DNA sensors is their ability to bind viral DNA through the HIN domain and procure protein-protein interactions via their pyrin domain, culminating in an innate immune response. Currently, IFI16 is the only known nuclear sensor. Importantly, recent work in the Cristea laboratory revealed that the interferon-inducible protein X (IFIX), another PYHIN protein, can recognize viral DNA in both the nucleus and the cytoplasm. Moreover, in uninfected cells we've found that IFIX interacts and co-localizes with promylocytic leukemia PML bodies, known to be involved in DNA repair and intrinsic immunity. Several PML body components have been shown to have antiviral roles that are inhibited by the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein ICP0. I have also found that viral proteins that are known to be involved in apoptosis inhibition interact with IFIX during infection. The goal of this proposal is o define the role of IFIX as a critical antiviral factor and characterize cellular pathways required or nuclear sensing of pathogenic DNA. I will use a multidisciplinary approach that integrates virology, proteomics, and bioinformatics to uncover the roles of IFIX during infection with HSV-1, a prominent human pathogen. First, I will establish that IFIX functions as a nuclear DNA sensor using IFIX localization-constricted mutants in conjunction with fluorescence in situ hybridization and measurement of antiviral cytokine levels. Second, I will identify and functionally characterize the IFIX association with PML body components during infection. Third, I will specifically characterize the subset of IFIX-viral protein interactions that lead to the observed IFIX- dependent reduction of HSV-1 progeny titers. Altogether, the proposed research will provide the first insights into the antiviral functions of IFIX during infection, bringing to light cellular evnts that culminate in immune deficiency and autoimmune diseases.